1. MICROBIAL BIOCHEMISTRY BIOT 309, 2012 Kim and Gadd, Chapter 4

2. OVERVIEW OF BACTERIAL METABOLISM

3. BACTERIAL METABOLISM
Metabolism = all biochemical reactions taking place in organism
Conversion (change, rearrangement) reactions
One molecule becomes another
Structure changes
Not use or generate energy
Anabolism uses endergonic reactions
Uses energy
Forms bonds
builds larger molecules, ie, proteins, carbohydrates
Also have passive transportmetabolism refers to the sum of the biochemical reactions required for energy generation AND the use of energy to synthesize cell material from small molecules in the environment.

4. BACTERIAL METABOLISM Catabolism is exergonic
Releases/produces energy, i.e., makes ATP
Breaks bonds
Hydrolyzes larger molecules into simpler molecules
Start here

5. COMPARISON Anabolism Catabolism Buildup of bigger molecules
Breakdown of larger molecules
Products are large molecules
Products are small molecules
Protein, peptidoglycan, DNA, RNA
Glycolysis, citric acid cycle
Mediated by enzymes
E required (endergonic)
E released (exergonic)
How many ATPS

6. BACTERIAL METABOLISM Growth depends on metabolism
All 3 types of reactions happening at the same time but anabolism or catabolism dominate at different phases of growth
Carried out by wide variety of enzymes and co-factors
Involves single enzymes and enzyme complexes
Provides precursor metabolites to anabolic pathways
Occurs in three locations:
Cytosol
On or in cell membrane
In periplasmic space
Certain phases primarily anabolic e.g., log phase
Catabolic: stationary phase
Talk about enzymes quick review
Mostly seen complexes to date – flagella, ATP synthase, electron transport, will revisit these during next 2 weeks

7. ENZYMES Characteristics Reusable Very specific – one reaction/enzyme
Minute amounts needed
Work fast ( reactions/minute)
Catalysts
Large proteins
Cont’d

8. ENZYMES Characteristics 1 enzyme/reaction Substrate specificity
“Lock and Key”

9. “LOCK AND KEY”

10. “LOCK AND KEY” with coenzyme*
* See descriptions in White * *

11. ENZYMES
Active site aligns substrate(s) so reaction is highly favorable
Free energy = G

12. ENZYMES Primarily proteins
Some have co-factors; co-factor use based on needs of enzyme
Inorganic ions: Mg++, Fe++, Zn++
Organic ions: important in redox reactions
NAD+ : EMP glycolysis, ED – Entner Duodorff pathway
NADP+: HMP – hexose monophosphate pathway, glycolysis
Both inorganic and organic used in reactions in TCA cycle and ETC (electron transport)

13. NAD+ (oxidized) NADH (reduced)
NAD in the oxidized form contains the elements of ADP, with an additional ribose molecule and a nicotinamide ring. The latter is the critical element in redox reactions. When a pair of H atoms (2 protons + 2 electrons) are removed from organic substrates in an oxidation reaction, NAD+ accepts 2 electrons and 1 proton; the remaining proton is released as free H+ ion.
As NAD+ is reduced, one electron is added at the Nitrogen atom (removing the + charge), (= becomes - ) and one (electron + proton = H atom) (= becomes -) is added at the upper position of the nicotinamide ring.

14. ENERGY STORAGE DURING CATABOLISM
Must be available as energy for anabolism
Forms of storage:
Held in high energy bonds, e.g., ATP
Reducing equivalents, such as NADH, NADPH & FADH2
Proton gradient (formed by electron transport system)
Forms used depend on pathway/enzymes used by bacteria
ATP and NADH are most common

15. COUPLED REACTIONS OCCUR: BE ABLE TO IDENTIFY THEM

16. Also Important in Glycolysis and Kreb’s Cycle!!!
Substrate-level phosphorylation
HOMEWORK: FIND EXAMPLES FROM SLIDES AND TEXTBOOK

17. ADDITIONAL REDOX MOLECULES
Used in Electron Transport – Ch 5
Ubiquinone
Iron-sulfur
Will review then

18. ENZYMES OCCUR AS: Single enzymes
Part of complexes with other proteins and cofactors *
Electron transport chain
Flagella
ATP synthase
Part of pathways
Glycolysis
Citric Acid Cycle
Etc.
Slowest reaction is a rate limiting step

19. BACTERIA: FOCUS ON CATABOLISM
Catabolism is exergonic
Releases/produces energy, i.e., makes ATP
Hydrolyzes larger molecules into simpler molecules
Breaks bonds
3 phases of catabolism: glycolysis, Kreb’s Cycle, Electron Transport Chain (ETC)
Also have passive transport

20. BIG PICTURE Integrating 3 Phases of Catabolism

21. Reaction Products have abbreviated names
Watch for their use and know to what they refer

22. GLYCOLYSIS Occurs mainly in cytoplasm
1st step in some bacteria occurs in membrane
Involves how many enzymes? TEN but two ways to make glucose-6-phosphate (See STEP 1 slide.)
Splits glucose
NOTE: Does not require O2, i.e., this stage is anaerobic

23. OVERALL GLYCOLYSIS* REACTION
glucose (6C) + 2 NAD+ + 2 ADP +2 Pi 2 pyruvate (3C) + 2 NADH + 2 H+ + 2 H2O + 2 net ATP Is NAD+ the oxidized or reduced form? *also called Embden-Myerhof-Parnas Pathway

24. GLYCOLYSIS AND ALTERNATIVES
Bacteria use 3 different pathways to convert glucose to PGA (3-phosphoglycerate) (see diagram)
Glycolysis/Embden-Myerhof-Parnas (shown in next slide)
Pentose phosphate shunt/hexose monophosphate shunt
Entner-Duodorff
Energy yields are different
Same pathway (transition or bridging reaction) takes PGA (3-phosphoglycerate) to pyruvate

25. GLYCOLYSIS 3 different glycolytic pathways operate: EMP, EDP, HMP
 THIS IS EMP
Divide in 2ne way = preparatory phase; other = payoff phase
From PGA on same steps

26. GLYCOLYSIS PHASES Preparatory Phase Payoff Phase
Divide in 2ne way = preparatory phase; other = payoff phase
Payoff Phase

27. HIGH ENERGY COMPOUNDS ATP Pyruvate
HOMEWORK: WHAT OTHER HIGH ENERGY COMPOUNDS ARE PART OF EMP PATHWAY?

28. Glucose glucose-6-phosphate (G6P)
GLYCOLYSIS, step 1
Rapid reaction to keep glucose inside cell
Location 1 = cytoplasm; one enzyme = hexokinase, requires Mg2+
Glucose glucose-6-phosphate (G6P)
Location 2: membrane (Some bacteria)
PEP pyruvate provides ~P to phosphorylate and transport glucose across the membrane
More proteins and enzymes are involved
Other sugars use similar mechanism
phosoenolpyruvate: sugar phosphotransferase system (PTS)
PEP + SUGAR PYRUVATE + sugar-phosphate
In E.coli the PTS consists of two enzyme and a low molecular weight heat-stable protein (HPr)
WHAT DOES PEP stand for?

29. GLYCOLYSIS, step 1 Group Translocation – phosphotransferase system
When bacteria use the process of group translocation to transport glucose across their membrane, a high-energy phosphate group from phosphoenolpyruvate (PEP) is transferred to the glucose molecule to form glucose-6-phosphate.
A high-energy phosphate group from PEP is transferred to the glucose molecule to form glucose-6-phosphate.
The glucose-6-phosphate is transported across the membrane.
Once the glucose has been converted to glucose-6-phosphate and transported across the membrane, it can no longer be transported back out.

31. ANIMATIONS FOR GROUP TRANSLOCATION ANIMATION:

32. GLYCOLYSIS, step 2
Rearrangement/change reaction, requires Mg2+

33. GLYCOLYSIS, step 3 phosphorylation
NOTICE use of ATP
QUESTION: What type of reaction is this?

34. GLYCOLYSIS, step 4 cleavage
Aldose to ketose isomerization
Yield 2 G3P
END OF PREPARATORY PHASE
Aldol cleavage

35. GLYCOLYSIS, step 5 coupled oxidation + phosphorylation☐
QUESTION: Where is energy of NADH used? Where does it go?
~ = high
energy bond
Question: What is oxidized? What is reduced?

36. GLYCOLYSIS, step 6 dephosphorylation
2 ADP goes to high energy 2 ATP as ~Pi removed from top
SLP involves soluble enzymes and chemical intermediates  ATP
Membrane bound enzymes and transmembrane gradients of proton s
Example of substrate level phosphorylation
QUESTION: From what carbon atom is the Pi removed?
Why is this Pi removed?

37. GLYCOLYSIS, step 7 phosphate group shift

38. GLYCOLYSIS, step 8 dehydration
~ = high energy bond
QUESTION: why does this reaction create ~Pi?

39. GLYCOLYSIS, step 9 dephosphorylation
~ = high energy bond

41. OVERALL GLYCOLYSIS* REACTION
glucose (6C) + 2 NAD+ + 2 ADP +2 Pi 2 pyruvate (3C) + 2 NADH + 2 H+ + 2 net ATP + 2H2O WHICH is oxidized and which is reduced? NAD+ is __________ ; NADH is ___________ *also called Embden-Myerhof-Parnas Pathway

42. THE OTHER GLYCOLYTIC PATHWAYS

43. TRANSITION OR BRIDGING REACTION Connects glycolysis to citric acid/Kreb’s Cycle
OVERALL REACTION 2 pyruvate + 2 NAD+ + 2 CoA-SH (coenzyme A) 2 acetyl-CoA + 2 NADH + 2 H+ + 2 CO2
CONNECTION TO OTHER BIOLOGY: Where else is CO2 made?

44. NAD+ (oxidized) NADH (reduced)
NAD in the oxidized form contains the elements of ADP, with an additional ribose molecule and a nicotinamide ring. The latter is the critical element in redox reactions. When a pair of H atoms (2 protons + 2 electrons) are removed from organic substrates in an oxidation reaction, NAD+ accepts 2 electrons and 1 proton; the remaining proton is released as free H+ ion.
Nicotinamide Adenine Dinucleotide
As NAD+ is reduced, one electron is added at the Nitrogen atom (removing the + charge), (= becomes - ) and one (electron + proton = H atom) (= becomes -) is added at the upper position of the nicotinamide ring.

45. ANOTHER COENZYME Coenzyme A
Energy generation
Molecule made from several component parts – complex
Highly polar
Key in glycolysis to Kreb’s cycle transition reaction
Key component in fatty acid reactions
Synthesis very similar pro- and eukaryotes

46. TRANSITION REACTION 3 carbon Co A 2 carbon
Before the pyruvates from glycolysis (see Fig. 2) can enter the citric acid cycle, they must undergo a transition reaction. The 3-carbon pyruvate is converted into a 2-carbon acetyl group with a carboxyl being removed as CO2. The acetyl group is attached to coenzyme A to form acetyl coenzyme A (acetyl-CoA), a key precursor metabolite. As the two acetyl groups become oxidized to acetyl-CoA, two molecules of NAD+ are reduced to 2NADH + 2H+.

48. NEXT: MORE ON TCA CYCLE

49. Operate under different growth conditions
Note energy yields

50. NOTE: MORE SLIDES WILL BE ADDED ON ED AND PPS PATHWAYS

51. ENTNER-DUDOROFF PATHWAY
The Entner-Doudoroff pathway yields one ATP and two NADPH molecules from one glucose molecule.
Uses totally different enzymes
1 Glucose pyruvate + 1 ATP + 1 NADH
+ 1 NADPH
Bacteria: Pseudomonas, Rhizobium, Azotobacter, Agrobacterium, Enterococcus faecalis

52. PENTOSE PHOSPHATE SHUNT PATHWAY
Precursors to the ribose and deoxyribose in nucleic acids
Provides erythrose phosphate which is a precursor for synthesis of aromatic amino acids
reducing power = NADPH
Overall reaction
6 Glucose 6 – P + 12 NADP H2O + 5 Glucose 6 – P +
6 CO NADPH + 12 H+
Used exclusively by Thiobacillus novellus and Brucella abortus

53. What do abbreviations stand for?
Pentose Phosphate
Shunt Pathway
What do abbreviations stand for?
MAKE a list!!!